Comparison of Antibacterial Effects of ZnO and CuO Nanoparticles Coated Brackets against Streptococcus Mutans.

STATEMENT OF THE PROBLEM
During the orthodontic treatment, microbial plaques may accumulate around the brackets and cause caries, especially in high-risk patients. Finding ways to eliminate this microbial plaque seems to be essential.


PURPOSE
The aim of this study was to compare the antibacterial effects of nano copper oxide (CuO) and nano zinc oxide (ZnO) coated brackets against Streptococcus mutans (S.mutans) in order to decrease the risk of caries around the orthodontic brackets during the treatment.


MATERIALS AND METHOD
Sixty brackets were coated with nanoparticles of ZnO (n=20), CuO (n=20) and CuO-ZnO (n=20). Twelve uncoated brackets constituted the control group. The brackets were bonded to the crowns of extracted premolars, sterilized and prepared for antimicrobial tests (S.mutans ATCC35668). The samples taken after 0, 2, 4, 6 and 24 hours were cultured on agar plates. Colonies were counted 24 hours after incubation. One-way ANOVA and Tukey tests were used for statistical analysis.


RESULTS
In CuO and CuO-ZnO coated brackets, no colony growth was seen after two hours. Between 0-6 hours, the mean colony counts were not significantly different between the ZnO and the control group (p>0.05). During 6-24 hours, the growth of S.mutans was significantly reduced by ZnO nanoparticles in comparison with the control group (p< 0.001). However, these bacteria were not totally eliminated.


CONCLUSION
CuO and ZnO-CuO nanoparticles coated brackets have better antimicrobial effect on S.mutans than ZnO coated brackets.


Introduction
White-spot lesions are the well-known side effect of fixed orthodontic treatment. During orthodontic treatment, increased proliferation of the facultative bacterial population, including S.mutans, leads to a decrease in pH which in turn can lead to development of white-spot lesions and eventually to cavitation and caries extending into the dentin. [1][2][3] Recently, nanotechnology has legitimated the  interfaces with other organic and inorganic molecules. [4] The antimicrobial properties of silver nanoparticle, known as the most common nanoparticle, are well recognized and numerous mechanisms for their bactericidal effects have been anticipated. [5][6][7][8] Silver ions are broadly employed as bactericides in catheters, burn wound care, and in the dental practice. [9] Although only a few studies have reported the antibacterial properties of copper and zinc nanoparticles, they have also shown copper and zinc nanoparticles to have a significant role as bactericidal agents. [4,[10][11] In this regard, Sierra et al. showed a higher antimicrobial effect against S.mutans of silver nanoparticles at lower concentrations than gold or zinc, which would permit attaining imperative clinical effects with lesser toxicity. [4] In another study, Ruparelia et  bial action of the silver nanoparticles was superior. [11] As there are concerns regarding the biological safety of silver nanoparticles (because of the closeness of size of nanosilver to silver ions) and due to the pigmentation effect of silver nanoparticles on teeth, [5][6][7] ZnO and CuO nanoparticles were used in this investigation. The purpose of this study was to examine the antimicrobial effects of coated brackets with ZnO and CuO nanoparticles against S.mutans.

Materials and Method
For preparation of CuO nanoparticles, aqueous solution of copper acetate (0.02 M) was prepared in round bottom flask. Then, 1 mL glacial acetic acid was added to the above-mentioned aqueous solution and heated to 100 º C with constant stirring. About 0.4 g of NaOH was added to the heated solution till pH reached 6-7.
A large amount of black precipitate was formed immediately. It was centrifuged and washed 3-4 times with de-ionized water. The obtained precipitate was dried in air for 24 hours. [12]In order to prepare ZnO nanoparticles, sodium hydroxide solution was added to the aqueous solution of zinc sulfate slowly dropwise in a ratio of 1:2 under vigorous stirring and the stirring was continued for 12 hours. The precipitate obtained was filtered and washed thoroughly with de-ionized water. The precipitate was dried in an oven at 100 º C and ground to fine powder. [13][14] The size and morphology of ZnO and CuO nanoparticles was examined using a transmission electron microscope (TEM; Philips CM200, Netherlands). According to the TEM evaluation, the mean diameter of ZnO and CuO nanoparticles was 45 and 37 nm, respectively ( Figure 1).In this study, spray pyrolysis was used for bracket coating. The spray pyrolysis is an excellent method for deposition of thin films of metallic oxides, in which, a starting solution, containing metal precursors is sprayed by means of a nozzle, assisted by a carrier gas, over a hot substrate. When the fine droplets reach the hot substrate, the solid compounds react on the surface and a new chemical compound is developed. It should be noted that this coating was only bonded to the metal surface. [15] The coating was approved by using   in the number of colonies in the interval of 0 to 2 hours, 4 to 6 hours, and 6 to 24 hours in different groups were compared by the One-way ANOVA ( Table 2). The results revealed a significant difference in the intervals of 0 to 2, 4 to 6, 6 to 24 hours; however, between 2 to 4 hours the mean difference in colony counts was not significant. The Tukey test showed that the mean differences were statistically significant among all the study groups except in ZnO-CuO group (p= 0.999), as well as in the ZnO and control groups (p=0.844) with an interval of 0 to 2 hours (Table 3). As illustrated in Table 4 ( Table 5)

Discussion
Nanotechnology has opened new trends in health. One of its most interesting characteristics is the antimicrobial feature that some nanoparticles demonstrate. [4][5]7] According to Table 1, the best findings were observed in group CuO and ZnO-CuO, which reduced the bacterial population to zero within two hours. The ZnO group, in comparison with the control group, was also effective in decreasing the number of S.mutans colonies but had less antimicrobial effects compared with the two former groups.
In the ZnO and control groups, we observed a decrease in the number of bacteria between 0 to 6 hours ( Table 2). This may be due to the reduction of nutrients as a result of the dilution of bacterial suspension; bacterial growth was not observed in the lag phase and as they continued to die naturally, the population of microorganisms decreased as time passed. [4][5] Between 6 to 24 hours, an increase in the bacterial population was observed in the control, as well as in ZnO groups, which may be related to the long interval of 18 hours between the two colony counts. However, the rate of bacterial growth was lower in the ZnO group because of its bacteriostatic characteristics. This may be due to the fact that the microorganisms had adapted to a new environment and their entrance into the logarithmic growth phase was probable.
Many studies examined the effects of different nanoparticles on various bacterial strains. [11,[18][19][20] One of the important mechanisms of the antimicrobial effects of nanoparticles is the destruction of the microorganism membrane. [5,[16][17] As Bacillus subtilis and S. mutans are both gram-positive bacteria, it may be expected that Cu nanoparticles may also affect S. mutans, as their anti-microbial effects on S. mutans were observed in our study.
In the study by Ruparelia et al., the antimicrobial effect of Cu and silver nanoparticles against different types of bacteria was investigated and they concluded t-  which was just as beautiful as steel brackets.
In the CuO and ZnO-CuO groups, the color of the brackets changed to copper, which was cosmetically unsuitable (Figure 3).
Considering that fact that brackets in the mouth are in contact with saliva, abrasions caused by a toothbrush and toothpaste and different types of foods and drinks with various temperatures and pHs, the authors suggest to evaluate the stability of the coated layers in the future studies.
Although the findings of this study on the antimicrobial effect of brackets in short-term were positive, other investigations should be carried out in order to prove our notion.

Conclusion
In the short term, the antimicrobial effect of coated brackets with CuO and ZnO-CuO nanoparticles on S.mutans was excellent since after almost two hours the number of bacteria were reduced to zero. The coated brackets with ZnO nanoparticles ranked second due to its antimicrobial effects which, although in comparison with the control group caused dramatic reduction in the number of mutans, it could not reduce the population of